The present invention more particularly relates to means for specifically controlling and regulating:                the angiogenesis of endothelial cells (EC), and        the contribution of NK and T cells to the regulation of the immune system.        
The different aspects of the invention share the common feature of implementing or requiring an anti-CD160 monoclonal antibody (mAb) of the present invention, namely the mAb referred to by the inventors as CL1-R2 (hybridoma Budapest Treaty deposit CNCM I-3204), or a conservative equivalent thereof.
The present invention indeed demonstrates that the receptor CD160 (previously also referred to as BY55), which is known to be expressed by cytotoxic NK and T subsets (CD56dim CD16bright CD3− NK; T CD8+; TCRγδ), is involved in both angiogenesis and immune system regulation. CD160 structure has been extensively described in prior art documents, see e.g. WO 98/21240 in the name of the DANA-FARBER CANCER INSTITUTE.
EC and Angiogenesis:
Angiogenesis, the formation of new capillaries from the preexisting blood vessels, is a crucial component of embryonic vascular development and differentiation, wound healing, and organ regeneration. It however also contributes to the progression of pathologies that depends on neovascularization, including tumor growth, diabetes, ischemic ocular diseases, and rheumatoid arthritis (Risau, 1997; Ferrara, 1997). While the most important mediators of angiogenesis, the vascular endothelial cell growth factor (VEGF) family and fibroblast growth factor family are well define, angiogenesis stands as a complex process involving multiple gene products expressed by different cell types all contributing to an integrated sequence of events.
WO 03/018048 in the name of ABTECH et al. relates to the use of two soluble HLA Class I molecules, namely sHLA-G1 and sHLA-B7, to inhibit angiogenesis or to detect angiogenic sites. Supportive to this anti-angiogenic effect is the demonstration that sHLA-G1 inhibits endothelial cells (EC) proliferation and migration. It is also shown that sHLA-G1 and sHLA-B7 may inhibit the progression of a tumor induced by grafting human prostate adenocarcinoma cells in nude mice.
WO 03/018048 also mentions that an anti-CD160 antibody referred to as CL1-R2 inhibits the action exerted by sHLA-G on EC migration. It is therefrom deduced that BY55 could be an endothelial receptor for sHLA-G (cf. WO 03/018048 as published, page 23 lines 3-8).
The skilled person would however notice that WO 03/018048 gives no publicly available source for the mentioned CL1-R2 antibody.
On the other hand, soluble HLA, such as sHLA-G1 and sHLA-B7, are natural ligands for numerous receptors.
Hence, there remains a need in prior art for means that would be sufficiently specific to the angiogenesis signaling pathways to enable the elucidation of the mechanisms they involve. Specificity is also needed to provide medically useful compounds that can exert a specific control on angiogenesis without necessarily disturbing other signaling pathways.
NK and T Cells and the Immune System:
NK cells constitute a subset of lymphocytes that play a role in innate immunity directed against virally-infected or tumor cells. Their effector functions are the killing of target cells and cytokine production. NK cells use a combination of inhibitory and activating receptors expressed at their cell surface to mediate target cell killing and cytokine release upon interaction with specific ligands. Upon specific engagement with these ligands present on target cells, they release cytolytic granules containing perforin and granzyme that contribute to target cell apoptosis. Upon contact with sensitive target cells, they also produce a number of cytokines, including IFN-γ, TNF-α and GM-CSF early in the innate immune response that modulate adaptive immunity by regulating T cell function. The release of IFN-γ by NK cells in both inflamed tissues and secondary lymphoid organs influence the dendritic cells-initiated adaptive immune response. IFN-γ secreted by uterine NK cells may also control placental development and vascularisation during pregnancy.
Yet, only few human activating NK cell receptors have been shown to induce cytokine production upon specific engagement.
KIR2□L4 (CD158d) induces IFN-γ, production in resting and activated NK cells. CD16 is a low-affinity FcγRIII receptor responsible for Ab-dependent cellular cytotoxicity (ADCC). Signaling via CD16 triggers the production of cytokines, including IFN-γ, GM-CSF, and several chemokines. Incubation of activated NK cells with anti-NKp30 or anti-NKp46 mAb led to IFN-γ production by NK cells. Human NKG2D activating receptor that recognizes the stress-induced MICA and MICB molecules as well as the ULBP family of molecules and plays a major role in NK cell-mediated cytotoxicity is apparently unable to produce cytokines once triggered by specific mAbs.
T cells including CD8+ and CD4+ T cells also produce cytokines. Th1 cells produce IL-2 and IFNγ, whereas Th2 cells produce IL-4. The effector functions of CD8+ T cells partially overlap those of CD4+ T cells. Naïve T cells can differentiate into at least two subsets with distinct cytokine patterns: T-cytotoxic 1 cells secrete a Th1-like cytokine pattern, while T cytotoxic 2 cells secrete Th2 cytokines. Currently, it is customary to consider IFN-γ to represent a typical type 1 cytokine, whereas the signature cytokine of type 2 response is IL-4.
Cytokines intervene in the differentiation and stimulation of antibody-producing B cell clones and the cytopathic action of cytotoxic T cells Likewise, cytokine secretion influences the cell-destroying capacity of NK cells, and the capacity of macrophages to phagocytose different bacterial plaque components.
The present invention provides with means for specifically controlling up- or down-regulation of cytokine production. The means specifically acts on the CD160 signaling pathways.
Among the different activating NK cell receptors described to date, CD160 is the only non-clonally expressed receptor on the majority of circulating NK cells. CD160+ cells correspond to the non-proliferating, highly cytolytic, CD56dim CD16+ NK subset. CD160 engagement by HLA-C molecules mediates cytotoxic function.
CD160 is expressed by circulating CD56dim CD16bright CD3− NK, which constitute the majority of PB-NK cells.
CD56dim NK cell subset is more naturally cytotoxic and produces less abundant cytokines than CD56bright subset following activation by monocytes. CD56dim NK cell subset also expresses a specific pattern of chemokine receptors and adhesion molecules. Such phenotype is characteristic of terminally differentiated effector cells. CD160+ NK cells have a high cytotoxic activity potential, do not proliferate to IL-2, and mediate cell lysis upon interaction with HLA-C.
In contrast to other human NK cell receptors described to date, CD160 receptor appears unique for the following reasons. It is encoded by a gene located on human chromosome 1, it is a glycosyl phosphatydil inositol (GPI)-anchored molecule and its cell surface expression is down-modulated by NK cell activation mediated by cytokines including IL-2 and IL-15. As described for the killer cell Ig-like inhibitory receptors, CD160 is also expressed by γδ T cells, and a subset of αβ CD8+ T cell.